Hoxy-benzamido-butyric acid

ABSTRACT

THE TREATMENT OF CERTAIN DISEASES OF THE CENTRAL NERVOUS SYSTEM WTH 3,4,5-TRIMETHOXYBENZAMIDO-BUTYRIC ACID AND PHARMACEUTICALLY-ACCEPTABLE SALTS THEREOF IS DISCLOSED.

United States Patent 3,808,337 TREATMENT OF DISEASES OF THE CENTRAL NERVOUS SYSTEM WITH 3,4,5-TRIMETHOXY- BENZAMIDO-BUTYRIC ACID William Ferrari, Modena, Italy, assignor to Instituto Chemioterapico Italiano S.p.A., Milan, Italy No Drawing. Filed June 29, 1972, Ser. No. 267,613 Int. Cl. A61k 27/00 U.S. Cl. 424-319 4 Claims ABSTRACT OF THE DISCLOSURE The treatment of certain diseases of the central nervous system with 3,4,5-trimethoxybenzamido-butyric acid and pharmaceutically-acceptable salts thereof is disclosed.

The present invention relates to a method of depressing the activity of the central nervous system of a living animal body and, more particularly, relates to a method of treating all types of Parkinsonism and other disorders of the central nervous system which respond to anticholinergic agents, with 3,4,5-trimethoxybenzamidobutyric acid or pharmaceutically-acceptable salts thereof.

Agents with anticholinergic activity act on the central nervous system of living animals by depressing the activity of acetylcholine. Such agents are useful in treating symptoms of disorders of the central nervous system in living animals such as spasm of the type induced by acetylcholine, for example, various kinds of epilepsy, Parkin sonism, and the like. The degree of utilization of anticholinergic agents is restricted by the undesirable sideeffects which they may concurrently produce. It is, therefore, highly desirable that an agent having anticholinergic activity affect the central nervous system without undue influence on the peripheral nervous system.

Employment of anticholinergic agents for the treatment of Parkinsonism is of particular interest. Parkinsonism is a condition involving disturbance of certain brain centers, such as the mesodiencephalic activating center of the brain stem, which causes muscular rigidity (catatonia) and may impair normal movements and the ability to speak and Write. It results from injury to basal ganglia and is frequently the sequel to virus-caused epidemic (lethargic) encephalitis. Symptoms include involuntary tremors, lack of facial expression, depressed emotional tone, and salivation. Compounds which control the symptoms of Parkinsonism are, in general, anticholinergic agents which are capable of passing the blood brain barrier. Atropine and extract from various species of Solanaceae have long been used in treating Parkinsonism, and, more recently, anticholinergic agents, such as trihexylphenidyl, procyclidine, ethopropazine, and diethazine have been so employed. These anticholinergic agents have met with limited success due to undesirable sideeifects which may result from their administration. Dryness of the mouth, blurred vision, mydriasis (enlargement of pupils), epigastric distress, nausea and constipation are examples of side-effects which may be caused by anticholinergic agents. The capacity to produce desired anticholinergic eifects without side-effects is a practical consideration in the evaluation of an agent employed in the treatment of disorders of the central nervous system.

The method of the present invention resides in the finding that the administration of 3,4,5-trimethoxybenzamidobutyric acid, preferably 'y-(3,4,5trimethoxybenzamido)-bntyric acid, which corresponds to the formula I OH; O

ice

and/or its pharmaceutically acceptable salts, is eflz'ective in treating disorders of the central nervous system of living animals which respond to anticholinergic agents. Preferably, the therapeutically-acceptable salts of the compounds of the present invention are water-soluble neutral salts and are preferably the sodium and potassium salts. Other suitable salts are, for example, the ammonium, magnesium or calcium salts of the free acid. Advantageously, the compounds of the present invention can be used to treat the symptoms of Parkinsonism and the like in man and lower mammals. Little, if any, undesirable side effects are observed when employing the method of this invention.

The compounds can be prepared by reacting 3,4,5-trimethoxybenzoyl chloride with aminobutyric acid at a temperature of about -5 to +5 C. The free amino acid is slurried in water and is neutralized with a hydroxylcontaining solution, e.g., sodium hydroxide solution. The mixture is chilled to within 5 C. to +5 C., and 3,4,5- trimethoxybenzoyl chloride is gradually added together with agitation, maintaining the temperature at below +5 C. The mole ratio of amino acid to the acid chloride is generally about 1 to 1.5 :1. The resulting solution is preferably treated with char to decolorize it; the char is separated, e.g., filtered; and the filtrate neutralized with dilute HCl or H 50 to about a Congo red indicator end-point. The resulting precipitate is separated, e.g., by filtration or centrifugation, washed with water, dried, then recrystallized from ethanol, separated by filtration, centrifugation or decantation and dried. The 3,4,S-trimethoxybenzoyl chloride is commercially available. The aminobutyric acid can be prepared by known methods, for example, by the HCl-catalyzed hydrolysis of the corresponding lactam, which is known in the art. The salts obtained by the hydrolysis can then be passed through an ion-exchange resin bed to obtain the free amino acid.

The pharmaceutically-acceptable salts of the free acid compound of the method of the present invention can be obtained in any convenient manner. For example, the free acid may be converted into the salts by reaction of the free acid with an appropriate base in the presence of an inert solvent. Appropriate bases to prepare the salt form of the compound are, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, magnesium hydroxide, and calcium hydroxide. The compounds employed in the method of the present invention are also described in United States patent application S.N. 50,949, filed June 29, 1970, now U.S. Pat. No. 3,697,563, in the name of Aldo Garzia.

In the method of treatment of disorders of the central nervous system which respond to anti-cholinergic agents, particularly Parkinsonism, in accordance with the present invention, the dosage of, for instance, 'y-(3,4,5-trirnethoxybenzamido)-butyric acid, which can be given, can vary widely within rather broad limits. The dosages generally range from about at least 1 mg./kg./day (milligrams per kilograms of body weight per day), preferably 1 to 500 mg./kg./day, more preferably 2 to 200 mg./kg./day, based on the free acid. Particularly beneficial results have been obtained using an amount in a range of about 2 to mg./ kg./ day, based upon the free acid. The treatment can consist of a single daily dose, or the above dosages can be given fractionally at periodic intervals, for example, two to four doses of about 5 to 50 mg./kg. can be administered per day.

Administration of 3,4,5 trimethoxybenzamido-butyric acid in accordance with the method of this invention can be oral or parenteral. When the compound utilized in the present invention is administered by parenteral injection, e.g., subcutaneous, percutaneous, intraperitoneal, intravenous, intraarterial, intracutaneous, intramuscular, or interarticular injection, it is preferably in the form of the corresponding water-soluble neutral salt, preferably the sodium salt.

In connection with oral administration, the compound is preferably administered as free acid but it can also be in the form of a pharmaceutically-acceptable salt, e.g., as

3,4,5 trimethoxybenzamido butyric acid and its low order of toxicity, as evidenced by tests in lower animals (representative of which are reported hereinafter), are indicative of utility in treating disorders of the central nervous system, such as Parkinsonism, in human beings the ammonium, sodium, potassium, magnesium or cal- 5 as well as in lower animals. cium salt. For oral administration, the active 7 (3,4,5- 'y (3,4,5 trimethoxybenzamido) butyric acid is trimethoxybenzamido)-butyric acid and a nontoxic pharevaluated for its efiectiveness against tremors produced maceutical carrier may, for example, take the form of in guinea pigs by 1,4 dipyrrolidino 2 butyne (trea pill, lozenge, tablet capsule, or a liquid suspension. Ex- 10 morine) utilizing a procedure which has been modified emplary carriers are solids, such as lactose, magnesium from that described by G. M. Everett et al., Science 124, stearate, calcium stearate, starch, terra alba, di- 79 (1956), a standard procedure for antitremorine screen. calcium phosphate, sucrose, talc, stearic acid, gelatin, In all the Examples, the amount of the sodium salt of agar, pectin, or acacia and liquids such as peanut oil, 7 (3,4,5 trimethoxybenzamido)-butyric acid adminissesame oil, olive oil, water or the like. tered is based on the free acid.

According to one suitable method, the free acid can EXAMPLE I be admlmstered mixed wlth a molar equivalent of sodium or potassium bicarbonate. Suitable tablets for human or Mlce of either sex am used and 'f mlce are animal use can be conveniently prepared generally conused each dosage e Each eh 1S glveh a chaltaining from about 5 to 500 mg. of 3,4,5 trimethoxy- 20 lengmg dose Of tremorme, 20 mllllgrarns p r kllograrn, benzamido-butyric acid, either as free acid or as a pharlntrapefltoneeuy one hour efter admlnlstrallon of h maceutically-acceptable salt thereof. The compounds of Sodium Salt Q 'Y (3,4,5 trlmethoXybehlemldo)"blltyfle the present invention are usually administered in prepaacide ammals are then obsehved for slgns Central rations containing about 0.01 to 75%, by weight, of 3,4,5- and P P effects 9 iremoflhe- 'Y-( e ytrimethoxybenzamido-butyric acid. For parenteral admin- Y{ acl'd p y for $Creen1ng y istration, a composition containing 3 4 5. i administering various sized dosages intraperltoneally mto amido-butyric acid may be in the form of a sterile solu- The animals- Table I gives a summary of the effective tion. Suitable preparations for parenteral injection pro- Hess 0f the treating agent With respect t0 tremors, motility, vide from about 5 to 500 mg. of 3,4,5-trimethoxy-benzand vagal symptoms in the animals produced by the adamido-butyric acid. In addition, the compound used in ministration of tremorine:

TABLE I Number of animals protected from- Amount Of 'Y-( ,4, rimethoxy- Number Tremors Motility symptoms Vagal symptoms benzamido)-butyrie acid of Tremorine administered (mg/kg.) animals (mg/kg.) Complete Partial Complete Partial Complete Partial 6 20 0 0 0 0 0 0 6 2o 3 3 3 0 o 6 6 20 a 3 4. 2 o 6 6 20 3 o o 6 0 6 6 20 2 0 0 0 0 0 6 20 1 0 0 0 o 0 6 20 0 o o 0 0 0 the method of the present invention, or compositions con- Since tremorine acts by virtue of its transformation to taining the same, may be either administered together with keto-tremorine, antagonists of it may act either at the or include other physiologically active materials and/or level of the central receptors or by hindering its transmedicaments, e.g., buffering agents, antacids, sedatives, formation to keto-tremorine. 'y (3,4,5-trimethoxybenztranquilizers, analgesics, or the like. amido)-butyric acid has been found to be active as an It will be understood that the compositions employed in antagonist of both keto tremorine and tremorine, thus the method of the present invention can be brought into indicating that it acts at the level of the central receptors. a unit dosage form by any suitable technique known to one skilled in the art. EXAMPLE H Desirably, a method of treatment of Parkinsonism Six mice of either sex are used as controls. To each minimizes as far as possible peripheral anticholinergic s dm ist d keto-tremorine in an amount of 1 g (e.g., antispasmodic) activity. One means of determining intraperitoneally. Each of the control specimens exhibits the possible peripheral eifects of a composition employed emol's, Catatonia and vagal Y P' I11 another group in treating, for instance, Parkinsonism is by observing its of Six mice, 10 g Of the Sodium Salt of 'Y-( effect upon acetylcholine produced spasm of an isolated tTimethOXYbeHZamidO)'butyrie acid is injected intraveileuln 3 4 5 trimethOXybenZamido buty1-ic acid does nously. One hour later, keto-tremorine in the amount of not oppose the effect of acetylcholine on an isolated ileum 1 is intraperitoneally injected into the mice- In of a guinea pig, even at dosages of 1000 mgJkg, oth five minutes, four m1ce exhlbit no tremors and the remethods for determining possible peripheral effects are mammg two exhlbl? tremors which are attenuated from by observing the response in an animal after injection the controls. The mice exhibit catatonia and vagal sympof acetylcholine and by observing the response of an toms; however, these are attenuated in comparison to the mal to stimulation of the peripheral stump of the vagal controlsnerve after treatment with the composition. In rats, 7- EXAMPLE IH tfimethoXybenzamido) iy acid does not Tremorine, when administered to an animal, has the the YP F F and y e effect of y eifect of lowering the animals temperature. Four groups choline which s intravenously administered or the effect of six mice each are employed to determine the effect of of the stimulatlon of the peripheral stump of the vagal tremorine and 'y (3,4,5 trimethoxybenzamido) butyric nerve. acid upon the rectal temperature of mice at a room tem- A chmcally useful substance for the treatment of Parkperature of 22:2 C. In a first group, a physiological msonism generally inhibits tremors which are produced solution is intraperitoneally injected. In a second group, in mice, rats, guinea pigs, and other laboratory animals tremorine in the amount of 20 mg./ kg. is intraperitonealthat have undergone treatment with tremorine. Tremorine 1y administered to each mouse. In a third group, the sodiserves as an acetylcholinemimetic for the neurons of the um salt of 'y (3,4,5 trimethoxybenzamido) butyric central nervous system. The higher order of activity of acid in the amount of 10 mf./kg. is administered intraperitoneally into each mouse, and in a fourth group, the sodium salt of 'y-(3,4,5 trimethoxybenzamido)-butyric acid in the amount of mg./kg. is administered intraperitoneally to each mouse, and one hour later, tremorine in the amount of 20 mg./kg. is administered intra- The above experiments show that the active compounds of the method of the invention antagonized the central eifect of the tremorine. Further, the amounts of 'y-(3,4,S- trimethoxybenzamido)-butyric acid, about 5 to 200 mg./

peritoneally to each mouse. Table II summarizes the 5 kg., which provide noticeable improved effects in antiresults.

TABLE II Rectal temperature of mouse at- Group Material administered 60 min. 0min. +30 min. +60 min. +90 min. +120 min I Physiologicalsolntion (IP) 36.4=!:0.16 36.4=|=0. 16 36.5i0. 19 36.3i0.20 36.310.15 11-- Tremorine 20 mg./kg., IP) 36.5:h0.13 29.11024 28.5 .l=0.46 29.5:b0.59 3005:0154 III -(3,4,fi-trimethoxybenzamido)butyric acid(10mg./kg.,IP) 36.2=|=0.16 35.6=|=0.17 35.3:e020 35.4;b0. 35.8=|=0.11 IV -(3, 4, 5-trlmethoxybenzamido)-butyric acid plus tremorlne (10 plus mg./kg.,IP) 36.355111 35.6i0.13 29.3=|=0.39 28.8i0.68 29.1=l;0.67 a0.1:|=0.53

EXAMPLE IV tremorine treatment, are well below the concentrations 7 (3,4,5 trimethoxybenzamido) butyric acid is unhzedm the toxlclty tests also useful in the treatment of tremors, hypomotility, and vagal symptoms which are created by a prior administra- 20 EXAMPLE VII non of 20 E trerPonPe mtfztpentoneauy In a clinical test, fifteen human patients sulfering from laboratory an1I nals; To mlce administered 20 various degrees of Parkinsonism are treated with 'y-(3,4,5- of tremorme mtrapqntoneauy and after .tremonne trimethoxybenzamido)-butyric acid. Intravenous dosages SymPtmPS occur F of 1- -F are 200 mg./day of the sodium salt of 'y-(3,4,5-trimethbenzam1do)-butyr1c acid s in ected mtrapentoneally 1n oxybenzamido) butyric acid based on the weight of the f amqlmt 9 10 mg/kg; l q f Tremors Ceae free acid, and oral dosages are 400 mg./day of 'y-(3,4,5- an 51X mlce hypomotlhty Improves of the 51x trimethoxybenzamido)-butyric acid. The active compound 'P g vagal symptoms are attenuated m each of the is administered to the patients for at least one month. SIX mice. Positive modifications of the extra ra 'd l t PY In! a Y P 01115, EXAMPLE V particularly tremors, of the Parkinsonism are witnessed. Th compound l d i h h d f h present Negative side effects due to the treatment are not observed. invention presents little, if any, toxicity risk. To groups It 15 clalmedi consisting of six mice, each is administered intraperitoneal- A {method f P t; Parklnsonism Comprising ly one of the following dosage amounts of the sodium mllllsterlng to a 11V1I1g anlmal y afflicted With nsalt of 'y (3,4,5 trimethoxybenzamido) butyric acid an i amount of 'Y-( tfimethoxybellin mg./kg.: 50, 100, 200, 400, 800, and 1600, Th i zam1do)-butyr1 c acid, or pharmaceutically-acceptable salt are observed for 72 hours and in none of the tests do any thereof, a mlxtlll'e 0f t foregoingdeaths occur, 2. The method of claim 1 wherein the pharmaceutical- XA 1 40 ly-acceptable salt is selected from the group consisting of Subacute toxicity tests are conducted on two, 6-mem zg potassium ammomum magnesium and calcium ber groups of rats and two, 6-member groups of guinea pigs. To one group of each type of animal is admin- The method of Clam} 1 amount of istered 200 of the Sodium Salt of (3,4,5 tr 1metho (ybenzam1do) -butyr1c acid or salt theremethoxybenzamido)-butyric acid intraperitoneally at the of admlmstered 1S m the range of about 1 to 500 mg/kgrate of one injection per day for 7 days. The remaining Per groups are subjected to a daily dose of 400 mg./kg. per A method of tl'eatlng y p Of r ers of the day, intraperitoneally administered, for 7 days. The central nervous System of a living animal y which animals are observed during the treatment days and for 0 responds to anticholinergic agents comprising administerseven days thereafter. No deaths are found to occur in 5 ing to the living animal body an effective amount of 3,4,5- any of the groups. Table HI provides 111 tabular form the trimethoxybenzamido butyric acid or pharmaceuticallyaverage y Welght 0f the sublects before treatment and acceptable salt thereof, or a mixture of the foregoing. then after treatment.

TABLE 111 References Cited 3;? Average gh UNITED STATES PATENTS Group Animal I? Initial Final 3,692,827 9/1972 Garzia 424....319

200 199.3=|=2 20914.7 40o 199,6 2 209:|:L9 STANLEY J. FRIEDMAN, Primary Examiner 200 596. 6=l=7.3 597. 316.3 400 596. 5=l;8.6 601.1:b7.9

1 Of 'y-(3,4,5-trimethoxybenzamido)-butyric acid. 

